Posted by
Guy Cox-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cy3-Cy5-registration-on-fluoview-1000-tp7579371p7579376.html
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Actually, Olympus is one of the very few companies who have released the curves of chromatic aberration of at least some of their objectives. Since this is, these days, absolutely critical information we should be putting pressure on all manufacturers to provide this as a datasheet with their objectives. They all have the data! To claim a lens as an achromat it needs to have two crossing points (where foci are the same) which are usually in the blue (~480nm) and the red (~650nm). The deviation between and beyond these points is not specified. It could easily be in the 430nm region at the midpoint between these wavelengths. However, I doubt if JP is using quite such a basic lens. A fluorite lens has the same two crossing points but a much lower deviation and should not give the depth difference JP sees. However, without a curve one cannot say much. An apochromat lens has three crossing points, and while these are traditionally red, green and blue the basic design principles allow any 3 points, and many makers now offer VC objectives corrected into the violet. There will still be deviations between the crossing points but they absolutely should not allow the focus shift JP is seeing.
When we buy a car it could cost as little as just one objective, and you would get quite a supercar for the price of a complete microscope. The car would come with comprehensive specification sheet. Why doesn't the microscope?
Guy
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Steffen Dietzel
Sent: Thursday, 29 November 2012 9:37 PM
To:
[hidden email]
Subject: Re: Cy3/Cy5 registration on fluoview 1000
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Hi Jean Pierre,
although .43 microns is a bit much, (if you were using oil immersion)
the phenomenon in general is normal. As Martin wrote, the chromatic
aberration is mainly caused by the objective. For example, we would find
that green (520 nm) and deep red (670 nm) were aligned pretty well, but
the orange channel might be off about 200 - 300 nm in z (and up to 70 nm
in xy). Color correction by the microscope optics works only so well,
and for a certain range. It's one of those things you would like to know
about your objective but the companies won't tell you. It also might
change over time. When using objectives in the infrared, I measured up
to 10 microns aberration in z.
For testing, we used fluorescent multicolor beads and compared intensity
centers in ImageJ. The beads don't need to be subresolution for this
purpose, 0.5 µm TetraSpeck (Molecular Probes) dried on a glass surface
do the trick, there are probably others. Be sure to embed them in the
same mounting medium you use for your samples. And you might want to
attach them to the coverslip *and* the slide and compare both, in case
it gets worse with depth.
Regards
Steffen
On 28.11.2012 23:26, Jean-Pierre CLAMME wrote:
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>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hi,
>
> I noticed that on our fluoview 1000, the Cy3 and Cy5 channels are off by
> one slice when doing (0.43 um/slice Z stacks). I can correct it by
> software but I was wondering if anyone else see that on their instrument
> and If I should /can get it fixed.
>
> Thank you
>
> JP
>
--
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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy
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